Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography–tandem mass spectrometry

Abstract

A confirmatory method has been developed and validated that allows for the simultaneous detection of medroxyprogesterone acetate (MPA), megestrol acetate (MGA), melengestrol acetate (MLA), chlormadinone acetate (CMA) and delmadinone acetate (DMA) in animal kidney fat using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The compounds were extracted from kidney fat using acetonitrile, defatted using a hexane wash and subsequent saponification. Extracts were then purified on Isolute™ CN solid-phase extraction cartridges and analysed by LC–MS/MS. The method was validated in animal kidney fat in accordance with the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCα) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 µg kg^–1, respectively, for MPA, MGA, MLA, DMA and CMA, with respective detection capability (CCβ) values of 0.20, 0.81, 0.68, 1.07 and 0.92 µg kg^–1. The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26% for MPA, MGA, MLA, DMA and CMA, respectively. Fortifying kidney fat samples (n = 18) in three separate assays showed the accuracy of the method to be between 98 and 100%. The precision of the method, expressed as % RSD, for within-laboratory reproducibility at three levels of fortification (1, 1.5 and 2 µg kg^–1 for MPA, 5, 7.5 and 10 µg kg^–1 for MGA, MLA, DMA and CMA) was less than 5% for all analytes.

Keywords:

• acetylgestagens
• kidney fat
• mass spectrometry
• decision limit
• detection capability

Acknowledgements

This work was funded by The State Laboratory, Co. Kildare, Ireland. The authors thank the staff at The State Laboratory for their practical assistance.